Regenerative medicine has entered a new phase in which stem cell populations are being transplanted into patients to restore damaged or diseased tissues such as liver and pancreas. Liver diseases, potentially leading to organ failure due to hepatitis viruses, alcohol consumption, diet and metabolic disorders, and other causes, constitute a major medical burden world-wide. Similarly, pancreatic conditions, particularly diabetes, are a leading cause of health problems and death world-wide. Stem/progenitor cell therapies represent possible approaches to address these needs for treatment, and clinical programs are expanding world-wide to explore these novel therapies further. Although many types of precursors are being tested for clinical programs treating liver and pancreas, only certain ones are possible for clinical programs in the near term.
Overview of Stem Cell Biology
The stem cells or their descendants, committed progenitors, are capable of both sustained proliferation and differentiation into specialized cells. The crucial defining distinction of stem cells is their ability to self-renew, i.e., to maintain indefinitely a population with identical properties, through either symmetric or asymmetric cell divisions. Progenitors, by contrast, serve a transitory role in the amplification of a cell population during development or regeneration. When the self-renewal capacity of precursors cannot be rigorously ascertained, investigators sometimes use the terminology “stem/progenitor cells”. The term is used also for cell therapies involving the use of both stem cells and/or progenitors.
Stem cells in the first stages of the developing mammalian embryo, along with primordial germ cells at later stages, have the remarkable capacity to give rise to all of the body's cell types, and are therefore termed pluripotent. Embryonic stem (ES) cells remain pluripotent during extensive expansion as established cell lines. The self-renewal potential of ES cells appears virtually unlimited, although the accumulation of spontaneous mutations and chromosomal rearrangements eventually degrades their practical utility. Similarly pluripotent stem cells can be generated through the reprogramming of mature somatic cells by the introduction of small sets of defined genetic factors, and the cells are termed induced pluripotent stem (iPS) cells.
Mesenchymal stem cells or MSCs can be derived from bone marrow, adipose tissue, umbilical cord tissue, Wharton's Jelly and amniotic fluid, grow readily in culture under ordinary culture conditions, can be transplanted by a vascular route or by grafting, and lineage restricted to any mesodermal fate (e.g., bone, cartilage, tendon, muscle). They are able to lineage restrict to endodermal or ectodermal fates but with exceedingly low efficiency, so much so that this feature is not of practical utility with respect to clinical programs. The usefulness of MSCs for clinical programs is proving to be primarily by their production of secreted paracrine signals (matrix and soluble factors) or by immune-modulatory mechanisms, findings that have resulted in their use in clinical programs world-wide to alleviate liver conditions and pancreatic conditions including diabetes.
TABLE 1Intrahepatic Lineage-dependent Phenotypic Traits in Human LiversMaturationalEarlyIntermediateLateLineage Stages(Stages 1-4; zone 1)(Stages 5-6; zone 2)(Stages 7-10; zone 3)Cell sizes7-9 μm-stem cells~20-25 μm~25-35 μm10-12 μm--hepatoblasts12-15 μm-committedprogenitors17-18 μm-adult cellsPloidyDiploidDiploid and withTetraploid or highersome tetraploid(depends on age ofperson)ProliferationHyperplastic growthHyperplastic growthHypertrophic growth(DNA synthesis withand with some(DNA synthesis withcytokinesis)hypertrophic growthnegligible cytokinesis)(depends on theextent ofcytokinesis)RepresentativeStem Cells: NCAM,Transferrin,P4503A41,genes expressedEpCAM, CD44H (no AFPTAT1,glutathione-S-and little to noFully regulatabletransferase1,albumin), CS-PGs1,4albumin2HP-PGs4Hepatoblasts:Factors associatedICAM-11, EpCAM, AFP1,with apoptosis1CD44H, constitutivealbumin2, P450A71,HS-PGs1,4Hepatocytes: enzymes inglycogen synthesis1, CX281, HS-PGs4, partiallyregulatable albumin2Levels of expression are due to lineage-dependent activation of transcription1, acquisition of relevant regulatory elements in transcription2, translational mechanism(s)3, posttranscriptional modifications (e.g., in Golgi)4AFP, alpha-fetoprotein; CD44, receptor for hyaluronans; CS-PG, chondroitin sulfate proteoglycan; CX, connexins (gap junction proteins); Cyp450, cytochrome P450s; HS-PG, heparan sulfate proteoglycan; ICAM-1, intercellular adhesion molecule-1; NCAM, neural cell adhesion molecule; TAT, tyrosine aminotransferase
Determined stem cells, commonly called “adult stem cells”, are in fetal and postnatal tissues but are restricted to specific lineages defined by a germ layer (ectoderm, mesoderm, endoderm). Determined stem cells (and their descendants, committed progenitors) replenish mature cells that are lost through normal turnover or injury and disease. Some mature cell types, such as blood cells and those lining the gut or the outer layer of the skin, have a limited lifespan and must be replaced rapidly. Other mature cells, such as cardiomyocytes and certain neurons, can persist for years. The proliferation and differentiation of stem cells must be regulated tightly to ensure life-long maintenance of appropriate numbers of specialized cells and of the stem cell compartment itself, under normal conditions and when cells are replaced because of disease or injury.
This invention provides a method for delivery of any stem cell population, most especially for determined stem cells or their committed progenitors, by targeting their delivery by direct injection or by grafting strategies to the reservoir of stem cell niches giving rise to liver and pancreas. For a discussion of grafting methods and “feeder effects” on stem cell cultures, see U.S. patent application Ser. Nos. 12/213,100 and 13/102,939, the disclosures of which are both incorporated in their entirety herein by reference.
Liver, biliary tree and pancreas are mid-gut endodermal organs central to handling glycogen and lipid metabolism, detoxification of xenobiotics, processing of nutrients for optimal utilization, regulation of energy needs, and synthesis of diverse factors ranging from coagulation proteins to carrier proteins (e.g., AFP, albumin, transferrin). The integrity of the body depends heavily on liver, biliary tree, and pancreatic functions, and failure in any of them, especially the liver, results in rapid death. In recent years it has become apparent that these tissues comprise maturational lineages of cells that are in epithelial-mesenchymal cell partnerships. Each lineage tree begins with an epithelial stem cell (e.g., hepatic stem cell) partnered with a mesenchymal stem cell (e.g., an angioblast).
These give rise to cellular descendants that mature coordinately. The maturational process generates epithelial and mesenchymal cells that change step-wise with respect to their morphology, ploidy, growth potential, biomarkers, gene expression and other phenotypic traits. The functions of the liver and of the pancreas are the net sum of phenotypic properties of all of the cells throughout the entire maturational lineages. In Table 1 we provide a representative example of this by summarizing phenotypic properties of parenchymal cells within the liver and at different maturational lineage stages. It is assumed that there are comparable lineage stages from stem cells or progenitors to mature cells and existing in the pancreas, but these have yet to be defined fully.
The pancreas is located retroperitoneally and provides digestive enzymes to the duodenum and hormones regulating metabolism. The organ is particularly sensitive to mechanical handling and has a propensity to release locally its enzymes leading to autolysis. This tendency has limited the types of surgery that can be done with this organ, including cell therapy for a pancreatic disease or condition. The liver is less sensitive to manual manipulation than the pancreas, but access to it requires abdominal surgery or laparoscopy or access through the biliary tree by endoscopy.
The present invention thus contemplates introducing cells to the liver and to pancreas without physically disturbing or compromising the physical integrity of these organs.